1. Field of the Invention
The present invention relates generally to the fields of cell physiology, neurology and neuro-oncology. More specifically, the present invention relates to a novel method of diagnosing and treating gliomas and meningiomas.
2. Description of the Related Art
Glial cells comprise a large proportion of the total cell population in the CNS. Unlike neurons, glial cells retain the ability to proliferate postnatally, and some glial cells still proliferate in the adult or aged brain. Uncontrolled glial proliferation can lead to aggressive primary intracranial tumors, the vast majority of which are astrocytomas, and therefore, of glial origin. Tumors of astrocytic origin vary widely in morphology and behavior, and, according to the 1993 WHO classification schema, can be separated into three subsets. Astrocytomas, the lowest grade tumors, are generally well-differentiated and tend to grow slowly. Anaplastic astrocytomas are characterized by increased cellularity, nuclear pleomorphism, and increased mitotic activity. They are intermediate grade tumors and show a tendency to progress to a more aggressive grade. Glioblastomas are considered the most aggressive, with poorly differentiated cells, vascular proliferation, and necrosis. Due to the common morphological heterogeneity of cells within a single tumor, such classification is not clear-cut and is somewhat unsatisfactory. The term xe2x80x9castrocyte-derived tumorsxe2x80x9d as used herein refers to astrocytomas. Meningiomas are tumor originating in the meninges.
Significant progress has been made in identifying physiologically important growth factors, receptors, and signal transduction pathways that control normal and malignant cell proliferation. It is now commonly accepted that growth factor binding leads to activation of oncogenes such as the ras/raf pathway, and ras in turn regulates gene expression through at least two mitogen-activated protein kinases. Interestingly, the ras/raf pathway is in crosstalk with the cAMP signaling cascade which is activated by numerous hormones and neurotransmitters.
Recent studies suggest that ion channels may function in regulating a cell""s proliferative ability. For example, mitogen-stimulated lymphocytes show an upregulation in the expression of a high conductance potassium channel (15). In murine fibroblasts, activation of the ras/raf signaling cascade induces expression of a Ca2+-activated K+ channel that appears to be essential in the cells"" proliferative response (17). The idea that ion channel expression may be necessary for cell cycle progression is also supported by observations that pharmacological blockade of ion channels can inhibit cell proliferation. This has been demonstrated in a number of cell types including melanoma (28), breast cancer cells (41), brown fat cells (30), and also in several glial cell types such as Schwann cells (5), retinal glial cells (32) and astrocytes (29).
Untransformed glial cells from which glial tumors may originate have been extensively characterized electrophysiologically (37). Surprisingly, they appear to be liberally endowed with voltage- and ligand-activated ion channels for Na+, K+, Ca2+ and possibly Clxe2x88x92 ions. It is generally assumed that these ion channels perform homeostatic roles in the brain and may facilitate maintainance of K+ and possibly Na+ and Clxe2x88x92 ion concentrations in the extracellular space. In contrast to the numerous reports on ion channel expression and activity in nonneoplastic glial cells, electrophysiological properties of astrocytoma cells and the potential role of ion channels in growth control of astrocytomas remain largely unexplored. Inwardly rectifying K+ currents have been demonstrated in several established astrocytoma cell lines (4).
Gliomas cells are a very heterogeneous cell population that lack common antigens. Consequently, the prior art is deficient in the lack of effective means of identifying and treating malignant gliomas. The present invention fulfills this longstanding need and desire in the art.
The present invention describes the expression of a chloride conductance with unique properties that selectively characterizes tumor-derived cells of glial origin. In the present invention, whole-cell patch-clamp techniques were used to characterize the biophysical and pharmacological properties of chloride channels in primary cultures and acutely isolated cells from biopsies of human astrocytomas and established cell lines. In all preparations, the expression of time-dependent and voltage-dependent outwardly rectifying currents was observed. These currents are sensitive to several Clxe2x88x92 channel blockers including chlorotoxin, a component of scorpion venom and also allow other anions to permeate. This chloride conductance is involved in the growth control of astrocytoma cells.
Expression of voltage activated ion channels was determined in primary cultures from 7 freshly resected human primary brain tumors and in a 7 established human astrocytoma cell lines. Astrocytoma cells consistently expressed voltage-dependent outwardly-rectifying currents. Currents activated at potentials greater than 45 mV and showed outward transients upon termination of voltage steps. Currents reversed at the Clxe2x88x92 equilibrium potential, suggesting that they were largely carried by Clxe2x88x92 ions. Altering [K+]o or [Na+]o did not alter currents; neither did replacement of [K+]i by Cs+ or [Na+]i by NMDG. Anion substitution experiments suggest the following permeability sequence, determined from shifts in tail current reversal potential: Ixe2x88x92 greater than NO3xe2x88x92 greater than Brxe2x88x92 greater than Clxe2x88x92 greater than acetate greater than isethionate greater than Fxe2x88x92 greater than glutamate. Currents were sensitive to the Clxe2x88x92 channel blockers chlorotoxin, DIDS, and DNDS, with chlorotoxin being most effective, yielding  greater than 80% block at 590 nM. DIDS (100 xcexcM) and DNDS (100 xcexcM) reduced currents by 33.5% and 38.2% respectively. Currents were also sensitive to zinc (100 xcexcM, 47% block) and cadmium (25 mM, 42% block). Reducing [Ca2+]o decreased outward currents by 58% and almost completely eliminated transients, suggesting that Clxe2x88x92 currents are Ca2 +-dependent. Clxe2x88x92 channel block resulted in altered cell proliferation as determined by 3H-thymidine incorporation, indicating that these channels are involved in astrocytoma growth control.
It is an object of the present invention to demonstrate that glial-derived tumor cells express a unique voltage-dependent Clxe2x88x92 channel which is not found in non-glial tumors, such as melanoma or breast carcinoma, nor in untransformed glial cells.
It is another object of the present invention to show that expression of this unique Clxe2x88x92 channel plays a role in the cells"" abnormal proliferative state.
It is yet another object of the present invention to demonstrate the sensitivity of glioma Clxe2x88x92 channels to chlorotoxin.
It is still another object of the present invention to provide a monoclonal antibody which specifically binds to glial-derived or meningioma-derived tumor cells.
It is still another object of the present invention to demonstrate that glioma cells can be targeted and/or eliminated by a recombinant chlorotoxin fused to a cytotoxic protein.
It is still another object of the present invention to provide a method to screen for malignant gliomas.
It is still yet another object of the present invention to provide a method of treating malignant gliomas, including glioblastoma multiforme and astrocytomas.
Thus, in accordance with the aforementioned objects, in one embodiment of the present invention, there is provided an antibody which specifically recognizes an antigen in chloride channels of glial-derived tumor cells.
In another embodiment of the present invention, there is provided a pharmaceutical composition, comprising a ligand which binds specifically to glial-derived or meningioma-derived tumor cells and a pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, there is provided a method of differentiating glial-derived or meningioma-derived neoplastic tumor tissue from non-neoplastic tissue, comprising the steps of: contacting a tissue of interest with an antibody that specifically recognizes an antigen in chloride channels of glial-derived tumor cells; and measuring the level of binding of the antibody, wherein a high level of binding is indicative that the tissue is neoplastic.
In still yet another embodiment of the present invention, there is provided a fusion protein, said protein comprised of: a ligand that specifically recognizes an antigen in chloride channels of glial-derived tumor fused to a cytotoxic moiety.
In still yet another embodiment of the present invention, there is provided a method of treating an individual having a glioma or meningioma, comprising the step of administering to said individual a pharmacologically effective dose of a composition of the present invention.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.